Background. RTX, an anti-CD20 monoclonal antibody, added to chemotherapy has proven to be effective in children and adolescents with high-grade, high-risk and matured non-Hodgkin lymphoma. RTX leads ...to prompt CD19+ B lymphocyte depletion. However, despite preserved immunoglobulin production by long-lived plasmablasts after treatment, patients remain at risk of prolonged hypogammaglobulinemia. Further, there are few general guidelines for immunology laboratories and clinical feature monitoring after B cell-targeted therapies. The aim of this paper is to describe B cell reconstitution and immunoglobulin levels after pediatric B-NHL protocols, that included a single RTX dose and to review the literature. Methods. A retrospective single-center study on the impact of a single RTX dose included in a chemotherapeutic pediatric B Non-Hodgkin Lymphoma (B-NHL) treatment protocols. Immunology laboratory and clinical features were evaluated over an eight hundred days follow-up (FU) period, after completing B-NHL treatment. Results. Nineteen patients (fifteen Burkitt lymphoma, three Diffuse large B cell lymphoma, and one Marginal zone B cell lymphoma) fulfilled the inclusion criteria. Initiation of B cell subset reconstitution occurred a median of three months after B-NHL treatment. Naïve and transitional B cells declined over the FU in contrast to the marginal zone and the switched memory B cell increase. The percentage of patients with IgG, IgA, and IgM hypogammaglobulinemia declined consistently over the FU. Prolonged IgG hypogammaglobulinemia was detectable in 9%, IgM in 13%, and IgA in 25%. All revaccinated patients responded to protein-based vaccines by specific IgG antibody production increase. Following antibiotic prophylaxes, none of the patients with hypogammaglobulinemia manifested with either a severe or opportunistic infection course. Conclusion. The addition of a single RTX dose to the chemotherapeutic treatment protocols was not shown to increase the risk of developing secondary antibody deficiency in B-NHL pediatric patients. Observed prolonged hypogammaglobulinemia remained clinically silent. However interdisciplinary agreement on regular long-term immunology FU after anti-CD20 agent treatment is required.
In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated ...immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.
Major clinically relevant inflammatory events such as septic shock and severe COVID-19 trigger dynamic changes in the host immune system, presenting promising candidates for new biomarkers to improve ...precision diagnostics and patient stratification. Hepcidin, a master regulator of iron metabolism, has been intensively studied in many pathologies associated with immune system activation, however these data have never been compared to other clinical settings. Thus, we aimed to reveal the dynamics of iron regulation in various clinical settings and to determine the suitability of hepcidin and/or ferritin levels as biomarkers of inflammatory disease severity.
To investigate the overall predictive ability of hepcidin and ferritin, we enrolled the patients suffering with three different diagnoses - in detail 40 patients with COVID-19, 29 patients in septic shock and eight orthopedic patients who were compared to nine healthy donors and all cohorts to each other.
We showed that increased hepcidin levels reflect overall immune cell activation driven by intrinsic stimuli, without requiring direct involvement of infection vectors. Contrary to hepcidin, ferritin levels were more strongly boosted by pathogen-induced inflammation - in septic shock more than four-fold and in COVID-19 six-fold in comparison to sterile inflammation. We also defined the predictive capacity of hepcidin-to-ferritin ratio with AUC=0.79 and
= 0.03.
Our findings confirm that hepcidin is a potent marker of septic shock and other acute inflammation-associated pathologies and demonstrate the utility of the hepcidin-to-ferritin ratio as a predictor of mortality in septic shock, but not in COVID-19.
Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been ...implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.
Background
The
PLAUR
gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in ...truncated protein variants whose precise functions have not been elucidated yet. The
PLAUR
gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the
PLAUR
splicing pattern impact on HAE severity was tested.
Methods and results
The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation.
Conclusions
PLAUR
alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an
PLAUR
splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.
Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate ...in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity.
showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members
and
were downregulated, while other genes such as
, or long non-coding RNA
were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.
Purpose
Hereditary angioedema (HAE) is a rare disease caused by a C1 inhibitor (C1-INH) deficit. Clinically, HAE is manifested by repeated episodes of localized subcutaneous or submucosal oedema ...attacks. Managing HAE patients in pregnancy is challenging, since there are only limited data on the safety and efficacy of various therapeutic approaches.
Methods
We present our clinical experience treating acute HAE attacks during pregnancy in six consecutive patients.
Results
During the pregnancies, 79 HAE attacks occurred. The most frequent were abdominal 53 (67.1%) followed by peripheral 21 (26.6%), facial 10 (12.7%), and laryngeal 10 (12.7%) oedemas; 13 (16.5%) attacks were combined. Fifty (63.3%) attacks were treated with recombinant human C1-INH (rhC1-INH); 17 (21.5%) with plasma-derived, pasteurized, nanofiltered C1-INH (pnfC1-INH); 13 (16.5%) with icatibant; and 1 (1.3%) with plasma-derived, nanofiltered C1-INH (nfC1-INH). Treatment had to be repeated in 5 attacks (6.3%). All six deliveries (one caesarean section and five spontaneous vaginal deliveries) were complication free. All pregnancies went to the full term and the patients delivered healthy babies with a birth weight ranging from 2850 to 3690 g. No congenital abnormalities were detected in the neonates. No abortions occurred.
Conclusions
Our results show good C1-INH or icatibant treatment efficacy for HAE attacks in pregnancy. The treatment by the first drug used was effective in 93.7% of all attacks. In 6.3% of attacks, a second treatment had to be used. No adverse effects were observed.
A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating ...protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.
Abstract Common variable immunodeficiency (CVID), the most frequent primary antibody disorder, is characterized by hypogammaglobulinaemia and impaired antibody production. Poor vaccination response ...is essential for the diagnosis of CVID. Their under laying defects remain to be elucidated. Routine determination of antibody production in serum from CVID patients after vaccination and investigation of B cell function in vivo is complicated due to substitution therapy. Therefore we investigated antibody production on the B-cell level by ELISPOT and characterized changes in B-cell subpopulations in CVID patients, including plasmablasts, in peripheral blood by flow cytometry after vaccination for specification of the diagnosis. Thirty-seven CVID patients and eighty healthy volunteers were immunized with tetanus toxoid and pneumococcal polysaccharide vaccines. Specific antibody levels and B cell subpopulations were measured before vaccination and on day 7 after vaccination by ELISPOT assay and flow cytometry respectively. Of the thirty-seven well defined CVID patients studied, thirty lacked detectable spot forming cells producing specific IgG, IgA or IgM antibodies against employed vaccines and seven had only weak responses compared to controls. In the control group, an increase in circulating plasmablasts on day 7 post immunization corresponded with the appearance of antibody forming cells. In contrast, CVID patients failed to increase plasmablasts significantly in peripheral blood after antigen challenge. Our findings indicate that CVID patients have a block in terminal B-cell differentiation and that flow based assessment of plasmablasts in peripheral blood after vaccination serves as a surrogate diagnostic marker for assessing in vivo antibody responses in patients suspected to have CVID.
Neutrophils impact on processes preceding the formation of bradykinin, a major swelling mediator in hereditary angioedema (HAE), yet their potential role in HAE pathogenesis has not been sufficiently ...studied. We assessed the relative mRNA expression of 10 genes related to neutrophil activation using RNA extracted from the peripheral blood neutrophils of 23 HAE patients in a symptom-free period and 39 healthy donors. Increased relative mRNA expression levels of CD274, IL1B, IL1RN, IL8, MMP9, and TLR4, together with a lack in their mutual correlations detected in HAE patients compared to healthy controls, suggested a preactivated state and dysregulation of patients’ neutrophils. Patients’ neutrophil-alerted state was further supported by increased CD11b, decreased CD16 plasma membrane deposition, and increased relative CD274+ and CD87+ neutrophil counts, but not by increased neutrophil elastase or myeloperoxidase plasma levels. As CD274 mediates inhibitory signals to different immune cells, neutrophils were cocultured with T-cells/PBMC. The decrease in CD25+ and IFN-γ+ T-cell/PBMC ratio in patients indicated the patients’ neutrophil suppressive functions. In summary, the results showed neutrophils’ alerted state and dysregulation at the transcript level in patients with HAE types I and II even in a symptom-free period, which might make them more susceptible to edema formation. Neutrophils’ T-cell suppressive capacity in HAE patients needs to be further investigated.
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